Preconditioning of human skeletal myoblast with stromal cell-derived factor-1α promotes cytoprotective effects against oxidative and anoxic stress

Ibrahim Elmadbouh, Husnain Kh Haider, Muhammad Ashraf, Juan Carlos Chachques

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Background and Objectives: The incidence of human autologous transplanted skeletal myoblast (SkM) cell death in ischemic myocardium was higher in the first few days after cell therapy. We proposed that human SkM treated by human stromal cell-derived factor (SDF-1α) protein or tranfected by SDF-1α, precondition them against oxidative or anoxic injury. Methods and Results: The purification of human SkM (80~90%) culture was assessed for desmin and CXCR4 expression using immunostaining and flow cytometry respectively. Cells were transfected to overexpress SDF-1α or treated with rSDF-1α (10~200 ng/ml, 1~4 h) were either exposed to anoxia or treated with 100μM H2O2 for different time periods (1~6 h anoxia) (1~3 h H2O2). Optimized conditions for transfection of SDF-1α gene into human SkM were achieved, using FuGeneTM6/phSDF-1α (3:2 v/w, 4 h transfection) with 125μM ZnCl2 (p<0.001), up to 7 days post-transfection as compared with transfected SkM without ZnCl2 and non-transfected control cells. Transfection efficiency was assessed by immunostaining, ELISA, western blots and PCR. LDH analysis showed significant decrease in release of LDH after exposure to 6 h anoxia or 100μM H2O2 for 2 h as compared with the normal un-treated or un-transfected SkM (p<0.001). In western blots assay, SDF-1α over-expressing human SkM or treated with rSDF-1 α induced marked expression of total Akt (1.2-fold) and phospho-Akt (2.7-fold), Bcl2 (1.6-fold) and VEGF (5.8-fold) after exposure to 6 h anoxia as compared with human SkM controls. Conclusions: The preconditioning of donor transplanted human SkM with SDF-1α increased cell survival and promoted cytoprotective effect against oxidative or anoxic injury that may be an innovative approach for clinical application.

Original languageEnglish (US)
Pages (from-to)50-60
Number of pages11
JournalInternational Journal of Stem Cells
Volume4
Issue number1
DOIs
StatePublished - Jan 1 2011
Externally publishedYes

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Skeletal Myoblasts
Chemokine CXCL12
Oxidative Stress
Transfection
Western Blotting
Desmin
Wounds and Injuries
Cell- and Tissue-Based Therapy
Vascular Endothelial Growth Factor A
Cell Survival
Myocardium
Flow Cytometry
Cell Death
Enzyme-Linked Immunosorbent Assay

Keywords

  • Anoxia
  • Apoptosis
  • Cytokine
  • SDF-1α
  • Skeletal myoblast

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

Cite this

Preconditioning of human skeletal myoblast with stromal cell-derived factor-1α promotes cytoprotective effects against oxidative and anoxic stress. / Elmadbouh, Ibrahim; Haider, Husnain Kh; Ashraf, Muhammad; Chachques, Juan Carlos.

In: International Journal of Stem Cells, Vol. 4, No. 1, 01.01.2011, p. 50-60.

Research output: Contribution to journalArticle

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abstract = "Background and Objectives: The incidence of human autologous transplanted skeletal myoblast (SkM) cell death in ischemic myocardium was higher in the first few days after cell therapy. We proposed that human SkM treated by human stromal cell-derived factor (SDF-1α) protein or tranfected by SDF-1α, precondition them against oxidative or anoxic injury. Methods and Results: The purification of human SkM (80~90{\%}) culture was assessed for desmin and CXCR4 expression using immunostaining and flow cytometry respectively. Cells were transfected to overexpress SDF-1α or treated with rSDF-1α (10~200 ng/ml, 1~4 h) were either exposed to anoxia or treated with 100μM H2O2 for different time periods (1~6 h anoxia) (1~3 h H2O2). Optimized conditions for transfection of SDF-1α gene into human SkM were achieved, using FuGeneTM6/phSDF-1α (3:2 v/w, 4 h transfection) with 125μM ZnCl2 (p<0.001), up to 7 days post-transfection as compared with transfected SkM without ZnCl2 and non-transfected control cells. Transfection efficiency was assessed by immunostaining, ELISA, western blots and PCR. LDH analysis showed significant decrease in release of LDH after exposure to 6 h anoxia or 100μM H2O2 for 2 h as compared with the normal un-treated or un-transfected SkM (p<0.001). In western blots assay, SDF-1α over-expressing human SkM or treated with rSDF-1 α induced marked expression of total Akt (1.2-fold) and phospho-Akt (2.7-fold), Bcl2 (1.6-fold) and VEGF (5.8-fold) after exposure to 6 h anoxia as compared with human SkM controls. Conclusions: The preconditioning of donor transplanted human SkM with SDF-1α increased cell survival and promoted cytoprotective effect against oxidative or anoxic injury that may be an innovative approach for clinical application.",
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AU - Chachques, Juan Carlos

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N2 - Background and Objectives: The incidence of human autologous transplanted skeletal myoblast (SkM) cell death in ischemic myocardium was higher in the first few days after cell therapy. We proposed that human SkM treated by human stromal cell-derived factor (SDF-1α) protein or tranfected by SDF-1α, precondition them against oxidative or anoxic injury. Methods and Results: The purification of human SkM (80~90%) culture was assessed for desmin and CXCR4 expression using immunostaining and flow cytometry respectively. Cells were transfected to overexpress SDF-1α or treated with rSDF-1α (10~200 ng/ml, 1~4 h) were either exposed to anoxia or treated with 100μM H2O2 for different time periods (1~6 h anoxia) (1~3 h H2O2). Optimized conditions for transfection of SDF-1α gene into human SkM were achieved, using FuGeneTM6/phSDF-1α (3:2 v/w, 4 h transfection) with 125μM ZnCl2 (p<0.001), up to 7 days post-transfection as compared with transfected SkM without ZnCl2 and non-transfected control cells. Transfection efficiency was assessed by immunostaining, ELISA, western blots and PCR. LDH analysis showed significant decrease in release of LDH after exposure to 6 h anoxia or 100μM H2O2 for 2 h as compared with the normal un-treated or un-transfected SkM (p<0.001). In western blots assay, SDF-1α over-expressing human SkM or treated with rSDF-1 α induced marked expression of total Akt (1.2-fold) and phospho-Akt (2.7-fold), Bcl2 (1.6-fold) and VEGF (5.8-fold) after exposure to 6 h anoxia as compared with human SkM controls. Conclusions: The preconditioning of donor transplanted human SkM with SDF-1α increased cell survival and promoted cytoprotective effect against oxidative or anoxic injury that may be an innovative approach for clinical application.

AB - Background and Objectives: The incidence of human autologous transplanted skeletal myoblast (SkM) cell death in ischemic myocardium was higher in the first few days after cell therapy. We proposed that human SkM treated by human stromal cell-derived factor (SDF-1α) protein or tranfected by SDF-1α, precondition them against oxidative or anoxic injury. Methods and Results: The purification of human SkM (80~90%) culture was assessed for desmin and CXCR4 expression using immunostaining and flow cytometry respectively. Cells were transfected to overexpress SDF-1α or treated with rSDF-1α (10~200 ng/ml, 1~4 h) were either exposed to anoxia or treated with 100μM H2O2 for different time periods (1~6 h anoxia) (1~3 h H2O2). Optimized conditions for transfection of SDF-1α gene into human SkM were achieved, using FuGeneTM6/phSDF-1α (3:2 v/w, 4 h transfection) with 125μM ZnCl2 (p<0.001), up to 7 days post-transfection as compared with transfected SkM without ZnCl2 and non-transfected control cells. Transfection efficiency was assessed by immunostaining, ELISA, western blots and PCR. LDH analysis showed significant decrease in release of LDH after exposure to 6 h anoxia or 100μM H2O2 for 2 h as compared with the normal un-treated or un-transfected SkM (p<0.001). In western blots assay, SDF-1α over-expressing human SkM or treated with rSDF-1 α induced marked expression of total Akt (1.2-fold) and phospho-Akt (2.7-fold), Bcl2 (1.6-fold) and VEGF (5.8-fold) after exposure to 6 h anoxia as compared with human SkM controls. Conclusions: The preconditioning of donor transplanted human SkM with SDF-1α increased cell survival and promoted cytoprotective effect against oxidative or anoxic injury that may be an innovative approach for clinical application.

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